Ligand-induced native G-quadruplex stabilization impairs transcription initiation

Abstract

G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and G4s are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. Here, we establish a sensitive G4-CUT&Tag method for genome-wide profiling of native G4s with high resolution and specificity. We find that native G4 signals are cell type–specific and are associated with transcriptional regulatory elements carrying active epigenetic modifications. Drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation. In contrast, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. Moreover, ligand-induced G4 stabilization modulates chromatin states and impedes transcription initiation via inhibition of general transcription factors loading to promoters. Together, our study reveals a reciprocal genome-wide regulation between native G4 dynamics and gene transcription, which will deepen our understanding of G4 biology toward therapeutically targeting G4s in human diseases.

Genome-wide mapping of G-quadruplex structures with CUT&Tag

“…Validation with single-stranded nuclease pre-treatment demonstrates that the observed G4 CUT&Tag signals arise from regions where the DNA duplex is melted (Figure 4A ). During revisions of our manuscript, another study also reported development of G4 CUT&Tag in human cells, providing complementary validation for the robustness of the method ( 82 ).…”

Genomic G-quadruplex folding triggers a cytokine-mediated inflammatory feedback loop to aggravate inflammatory diseases

“…Furthermore, we verified that G4 stabilization could upregulate TNF-α and IL-6, and these cytokines could promote G4 stabilization in cells. We speculate that the folded G4 may regulate the expression of inflammatory genes by the direct interaction with DNA binding proteins such as transcription factors, modulating CpG methylation and shaping chromatin to produce an inflammatory transcriptome ( Lago et al., 2021 ; Li et al., 2021 ; Lyu et al., 2021 ; Tian et al., 2018 ). Therefore, we proposed that the crosstalk of G4 and inflammation may play a vital role in inflammation-derived diseases ( Figure 6 ).…”

“…Moreover, G4 has been recognized to play an important role in multiple key biological processes that involve replication, transcription, translation, genome instability, and telomere maintenance across multiple species ( Verma et al., 2008 ; Kwok and Merrick, 2017 ; Sengupta et al., 2021 ). In transcription regulation, G4 was found to facilitate the adherence of transcription initiation complex, increase promoter-proximal Pol II pausing, control CpG island methylation and interact with transcription factors ( Lago et al., 2021 ; Li et al., 2021 ; Lyu et al., 2021 ; Mao et al., 2018 ). Huang et al.…”

Modulating gene expression in breast cancer via DNA secondary structure and the CRISPR toolbox

“…This finding was consistent across two different cell lines with distinct G4 landscapes, suggesting that G4 binding is a general property of certain TFs ( 115 ). However, a recent study using the G4-CUT&Tag method demonstrated that ligand-induced G4-stabilization actually reduced transcriptional activity by inhibiting binding of TFs ( 116 ). Thus, while endogenous G4s have been previously associated with enhanced transcription, G4-stabilizing ligands may actually downregulate or suppress transcription ( 116 ).…”

“…However, a recent study using the G4-CUT&Tag method demonstrated that ligand-induced G4-stabilization actually reduced transcriptional activity by inhibiting binding of TFs ( 116 ). Thus, while endogenous G4s have been previously associated with enhanced transcription, G4-stabilizing ligands may actually downregulate or suppress transcription ( 116 ). Further investigation to see if this pattern is consistent over different ligand concentration, ligands, G4s and genes will be an interesting future direction.…”